Elsevier

Methods in Enzymology

Volume 143, 1987, Pages 459-465
Methods in Enzymology

l-Methionine γ-lyase from Pseudomonas putida and Aeromonas

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This chapter describes the assay method, purification procedures, and general properties of L-methionine γ -lyase from Pseudomonas putida (P. putida) and Aeromonas. L-Methionine γ-lyase catalyzes α, γ-elimination of L- methionine to produce α-ketobutyrate, methanethiol, and ammonia. The enzyme has been purified from Clostridium sporogenes, Pseudomonas putida and Aeromonas. The enzymatic α, γ-elimination of L-methionine can be routinely followed by spectrophotometric determination of α-ketobutyrate with 3-methyl-2-benzothiazolone hydrazone hydrochloride. In the case of preparation from P. putida, Pseudomonas putida ICR 3460 is grown in a medium containing 0.25% L-methionine, 0.1% polypeptone, 0.1% glycerol, 0.1% KH2PO4, 0.1% K2HPO4, 0.01% MgSO4·7H2O, and 0.025% yeast extract at 28° for 18 hr with shaking. The enzyme is crystallized by the vapor diffusion method of McPherson. L-Methionine γ-lyase is also purified from Aeromonas sp. ICR 3470 essentially by the same method as described for preparation from P. putida, using DEAE-Toyopearl 650 M and DE AE- Sephadex A-50 column chromatography. About 30 mg of a homogeneous preparation (specific activity, 31 units/mg) is obtained from a crude extract of 204 g of wet cells (total protein, 4.6 g; specific activity, 1.04 units/rag), with an overall yield of 20%.

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